chapter 9: catalytic strategies - sogang ocwocw.sogang.ac.kr/rfile/2014/biochemistry...

CHAPTER 9: CATALYTIC STRATEGIES

Chess vs Enzymes

King vs Substrate

INTRODUCTION

What are the sources of the catalytic power and specificity of

enzymes?

Problems in reactions in cells

• Neutral pH , water as a solvent and 37 ˚C

• Hard to achieve optimal reaction rate at these conditions

• Need a special strategy to achieve specificity

Enzyme classes discussed in this chapter

• Serine proteases

• Carbonic anhydrases

• Restriction endonucleases

• Myosins

CHAPTER 9

INTRODUCTION

Comparison between class members reveals how enzyme

active sites have evolved and been refined

Our knowledge of catalytic strategies has been used to

develop practical applications

• Potential drugs

• Enzyme inhibitors

• Other catalytic molecules (catalytic RNA)

CHAPTER 9

A FEW BASIC CATALYTIC PRINCIPLES

Substrate binding

• Establishes substrate specificity

• Stabilizes the transition state (lowers the activation E)

• Increases effective concentration

• Induced fit : the binding promotes structural changes that

facilitate catalysis

Covalent catalysis

• The active site contains a reactive group (nucleophile)

• The nucleophile becomes temporarily attached to a part of

the substrate in the course of catalysis

• Proteolytic enzymes

CHAPTER 9

A FEW BASIC CATALYTIC PRINCIPLES

General Acid-Base Catalysis

• A molecule other than water plays the role of a proton donor

or acceptor

• Histidines in chymotrypsin and carbonic anhydrase,

phosphate group of ATP in myosins

Catalysis by Approximation

• Reaction rate enhancement by proximity effect

Metal Ion Catalysis

• Nucleophile activation (carbonic anhydrase)

• Stabilizing reaction intermediate (EcoRV)

• Increasing binding affinity of substrates (myosin)

CHAPTER 9

9.1 CATALYSIS OF PROTEASES

Proteases facilitate a fundamentally difficult reaction

Involved in protein turnover

Important in regulating the activity of enzymes and proteins

Perform a hydrolysis reaction

• The addition of a water molecule to a peptide bond

• Thermodynamically favored reaction

• Half-life ~ 10 – 1000 yrs at physiological condition

• Peptide bonds need to be hydrolyzed within miliseconds in

some biochemical processes

CHAPTER 9

9.1 CATALYSIS OF PROTEASES

Kinetic stability of peptide bonds

• The resonance structure of a peptide bond

- Planar conformation

- Partial double bond character

- The amide carbon is less electrophilic than the ester carbon

• These properties make the peptide bond stable

CHAPTER 9

CHYMOTRYPSIN POSSESSES

A HIGHLY REACTIVE SERINE RESIDUE

Chymotrypsin cleaves peptide bonds selectively on the

carboxyl side of the large hydrophobic AA

• C-term of Trp, Phe, Tyr and Met

• A good example of the use of covalent catalysis

- A powerful Nu: attacks the unreactive carbonyl carbon of

the substrate

- The Nu: becomes covalently attached to the substrate

CHAPTER 9.1 PROTEASES

Fig 9.1 Specificity of chymotrypsin

Chymotrypsin cleaves the amide bond

of the carboxyl side of hydrophobic

amino acid.

CHYMOTRYPSIN POSSESSES

A HIGHLY REACTIVE SERINE RESIDUE

Identification of the reactive Nu: in chymotrypsin

• Treatment of chymotrypsin with diisopropylphospho-fluoridate

(DIPF) makes the enzyme irreversibly inactive

• Only a single residue, Ser195 was modified

- The residue plays a central role in the catalytic mechanism

of chymotrypsin


Fig 9.2 An unusually reactive serine

residue in chymotrypsin.

Chymotrypsin is inactivated by

treatment with DIPF, which reacts only

with Ser195 among 28 possible serine

residues

TWO-STEP ACTION OF CHYMOTRYPSIN

Kinetic study of chymotrypsin

• N-acetyl-L-phenylalanine p-nitrophenyl ester was used to

monitor the reaction by a colored product.


Fig 9.3 Chromogenic substrate.

N-acetyl-L-phenylalanine p-nitrophenyl ester

yields a yellow product, p-nitrophenolate, on

cleavage by chymotrypsin.


Kinetic study of chymotrypsin

• Michealis-Menten kinetics with a KM of 20 mM and a kcat of

77 s-1.

• Hydrolysis proceeds in two steps: an initial rapid burst

followed by a steady-state


Fig 9.4 Kinetics of chymotrypsin

catalysis. Two phases are evident in

the cleaving of N-acetyl-L-phenylalanine

p-nitrophenyl ester by chymotrypsin.


Explanation of the two-step hydrolysis

• The first step: acyl-enzyme intermediate formation

- Ser195 attacks the carbonyl carbon of the substrate

- p-nitrophenolate is released

• The second step: Hydrolysis of the acyl-enzyme intermediate


Fig 9.5 Covalent Catalysis. Hydrolysis by chymotrypsin takes place in two steps: (A) acylation to

form the acyl-enzyme intermediate followed by (B) deacylation to regenerate the free enzyme.

CATALYTIC TRIAD

The 3-D structure of chymotrypsin was solved in 1967

Comprises three polypeptide chains linked by disulfide bonds


Fig 9.6 The 3-D structure of chymotrypsin.

Synthesized as a

single polypeptide

(chymotrypsinogen),

which is activated by

the proteolytic

cleavage to yield the

three chains

CATALYTIC TRIAD

The active site lies in a left on the surface of the enzyme

Two hydrogen bonds in the active site

• Between Ser195 and His57

• Between His57 and Asp102

These three residues are referred to as the catalytic triad


Fig 9.7 The catalytic triad. The catalytic triad, shown on the left, converts serine 195

into a potent nucleophile, as illustrated on the right

CATALYTIC TRIAD


Fig 9.8 Peptide hydrolysis by chymotrypsin.

Substrate binding

Nucleophilic attack

Cleavage of the amide bond

Release of the amino

component

Water binding

Nucleophilic attack

Cleavage of the ester bond

Release of the carboxylic

acid component

Stabilizes the (-) charge of the

intermediate

Activate the Nu:

CATALYTIC TRIAD


Fig 9.11 Specificity nomenclature for protease-substrate interactions.

Fig 9.9 The oxyanion hole.Fig 9.10 Specificity pocket of

chymotrypsin

Positioning of Ser195

Hydrophobic environment

CATALYTIC TRIADS FOUND IN OTHER ENZYMES

Catalytic triads are found in other

hydrolytic enzymes

• Trypsin and elastase are obvious

homologs of chymotrypsin

- 40% identical sequence

- overall structures are quite similar

- remarkable difference in substrate

specificity: aromatic/hydrophobic,

long/(+) charged, and small (Fig

9.13)


Fig 9.12 Structural similarity of

trypsin and chymotrypsin.

Chymotrypsin (red); trypsin (blue)



Fig 9.13 The S1 pockets of chymotrypsin, trypsin, and elastase.



Fig 9.14 The catalytic triad and oxyanion hole of subtilisin.

Catalytic triads are found in many other hydrolytic enzymes

This catalytic strategy must be an especially effective approach

to the hydrolysis of peptides and related bonds

SITE-DIRECTED MUTAGENESIS STUDY


Fig 9.15 Site-directed mutagenesis of subtilisin.

How can we prove that the proposed mechanism is correct?

One way is to test the contribution of individual AA

Each residues within the catalytic triad in subtilisin are individually

converted into Ala

• Asp32, His64, and Ser221

• By site-directed mutagenesis

Oxyanion hole

• Asn155 to Gly

• kcat reduces to 0.2%

• Shows the significant role

of the amide proton

OTHER PEPTIDE-CLEAVING ENZYMES


Fig 9.16 Three classes of proteases

and their active sites.

Three alternative approaches to peptide-bond hydrolysis



Fig 9.17A The activation strategy for

cysteine proteases.

Cysteine proteases

• Their catalytic strategy is similar to the chymotrypsin family

• Cysteine plays the role of serine in chymotrypsin

• The cysteine is activated by a histidine residue

• Asp in the catalytic triad does not exist. (better nucleophilicity

of Cys)



Fig 9.17B The activation strategy for

aspartyl proteases.

Aspartyl proteases

• There are two aspartic acid residues in the active site

• One Asp activates the attacking water molecule

• The other polarizes the peptide carbonyl group

• Examples are renin and pepsin



Fig 9.17C The activation strategy for

metalloproteases.

Metalloproteases

• The active site contains a metal ion (zinc in most cases)

• The metal ion activates the attacking water molecule and

polarizes the peptide carbonyl group

• Examples are thermolysin and

carboxylpeptidase A

PROTEASE INHIBITORS


Figure Structures of captopril

Captopril

• The first angiotensin-converting enzyme inhibitor (ACE

inhibitor)

• Developed in 1975 by three researchers at the U.S. drug

company Squibb (now Bristol-Myers Squibb)

• Used for the treatment of

hypertension and some types of

congestive heart failure ( )

//upload.wikimedia.org/wikipedia/commons/0/04/Captopril-3D-vdW.png

//upload.wikimedia.org/wikipedia/commons/5/5f/Captopril.svg

PROTEASE INHIBITORS


Indinavir

• A protease inhibitor used to treat HIV infection and AIDS

• FDA-approved in 1996

• Aspartyl protease

• Non-peptidic substrate analog

▲Fig 9.18 HIV protease, a dimeric

aspartyl protease.

◄Fig 9.19 Indinavir, an HIV protease

inhibitor.

9.2 CATALYSIS OF CARBONIC ANHYDRASES

Hydration of carbon dioxide (CO2)

• CO2 is a major end product of aerobic metabolism and

released into the blood and transported to the lungs

• In the red blood cells, it reacts with water.

CHAPTER 9

pKa = 3.5

k1 = 0.0027 M-1s-1

k-1 = 50 s-1

K1 = 5.4 X 10-5

Carbonic anhydrases (CAs) accelerate CO2 hydration

• The most active CAs hydrate CO2 at kcat = 106 M-1s-1.

ZINC ION IN CARBONIC ANHYDRASE (CA)


CA was discovered in 1932

In 10 years after the discovery, the enzyme was found to

contain a bound zinc ion

• Appeared to be necessary for catalytic activity

• It made CA the first known zinc-containing enzyme

Hundreds of enzymes are known to contain zinc.

One-third of all enzymes contain or require metal ions for

activity

• Metal ions have several properties that increase chemical

reactivity: (+) charges, strong yet kinetically labile bonds, and

several oxidation states

• The chemical properties explain why they are important for

enzyme activity

ZINC ION IN CARBONIC ANHYDRASE (CA)


At least seven CAs are present in human beings

• They are all clearly homologous

• CA II is a major protein component of red blood cells and one

of the most active CAs

Fig 9.21 The structure of human CA II

and its zinc site.

CATALYSIS ENTAILS

ZINC ACTIVATION OF A WATER MOLECULE


pH dependence of enzymatic

CO2 hydration

• kcat increases with increasing pH

• The midpoint is near pH 7

Fig 9.22 Effect of pH on CA activity.

Fig 9.23 The pKa of zinc-bound water. Binding to zinc lowers the pKa of water form

15.7 to 7.

CATALYSIS ENTAILS



Zinc ion acts as a Lewis acid

and lowers the pKa of the bound

water

The zinc-bound OH- is a potent

nucleophile

CA also possesses a

hydrophobic patch

• Serves as a binding site for

carbon dioxide Fig 9.24 Carbon dioxide binding site.

CATALYSIS ENTAILS



Mechanism of CA

• Water deprotonation

• CO2 binding

• Nucleophilic attack by HO-

• Displacement of HCO3- by

water

Fig 9.25 Mechanism of CA.

CATALYSIS ENTAILS



Fig 9.26 A synthetic analog model system for carbonic anhydrase.

Studies of a synthetic analog model system

• Provide evidence for the mechanism’s plausibility

• The pKa of the bound water is 8.7

• At pH 9.2, this complex accelerates the hydration of CO2

more than 100-fold

• This experiment supports the proposed mechanism is

correct

PROTON TRANSFER IN CA CATALYSIS


Fig 9.27 Kinetics of water deprotonation.

Protons diffuse very rapidly with 2nd order rate constants

near 1011 M-1s-1.

k-1 ≤ 1011 M-1s-1 and K = 10-7 M, then k1 ≤ 104 s-1

If CO2 is hydrated at a rate of 106 s-1, then every step in the

mechanism must take place at least this fast!!

(pKa of the bound water is 7)



Fig 9.28 The effect of buffer on deprotonation.

The highest rates of carbon dioxide hydration require the

presence of buffer.

k1′ and k-1′ will be limited by buffer diffusion, ≤ 109 M-1s-1

At [B] = 1 mM, the rate of proton abstraction becomes

106 M-1s-1

K = k1′/k-1′ ≈ 1

if pKa of BH+ is 7.



Fig 9.29 Histidine proton shuttle.

His64 in CA II functions as the buffer

His64 abstracts a proton from the zinc-bound water and then

transfer the proton to the buffer

In many enzymatic reactions, the proton transfer is crucial to

the function

9.3 CATALYSIS OF RESTRICTION ENZYMES

Bacteria and archaea have mechanisms to protect

themselves from viral infections

• A major protective strategy for the host is to use restriction

endonucleases (REs)

REs recognize particular base sequences

REs must show tremendous specificity at two levels

• They must not degrade host DNA containing the recognition

sequences

• They must cleave only DNA molecules containing

recognition sites

For example, a recognition site, 5′-GATATC-3′, requires more

than 46 (4096) times more efficient activity

How do REs achieve these specificity?

CHAPTER 9

CLEAVAGE MECHANISM


Fig 9.32 Hydrolysis of a phosphodiester bond.

REs catalyze the hydrolysis of the phosphodiester backbone

of DNA

• Generate a free 3′-hydroxyl group and a 5′-phosphoryl

group

TWO PROPOSED MECHANISMS


Mechanism 1 (covalent intermediate)

Mechanism 2 (direct hydrolysis)



How can we figure out which mechanism is correct?

In-line displacement (SN2)

• Interconversion of the R and S configurations

Comparison of the two mechanisms

• In both cases, the reaction takes place by in-line displacement

• Two conversions occur in mechanism 1

• Only one conversion occurs in mechanism 2



How can we figure out which mechanism is correct?

Analysis of the product configuration

• A problem is that the product is not chiral !

Fig 9.33 Labeling with phosphorothioates.

Designing a special substrate

• Phosphorothioate

• Water containing 18O



Fig 9.34 Stereochemistry of cleaved DNA.

The analysis revealed that the stereochemical configuration

at the phosphorus atom was inverted only once with

cleavage

→ Mechanism 2

MAGNESIUM FOR CATALYTIC ACTIVITY


One or more Mg2+ are essential to the function of RE

• As many as three metal ions have been found to be present

per active site

• The roles of the multiple metal ions is still under investigation

Fig 9.35 A magnesium ion-binding site in EcoRV.

• One ion-binding site

appears in all RE

structures

• In the EcoRV structure,

the Mg2+ activates and

positions a water

molecule to attack the

phosphorus atom

THE ORIGIN OF THE SEQUENCE-SPECIFICITY


The recognition sequences for most REs are inverted repeats

• Palindromic sequence / twofold rotational symmetry

• Most REs functions as a dimer

Fig 9.36 Structure of the recognition site of EcoRV.



The binding affinity of EcoRV to the cognate DNA

• The enzyme can bind DNA in the absence of Mg2+

• Almost no affinity difference between the cognate and

noncognate !

Fig 9.37 Structure of EcoRV embracing a cognate

DNA molecule.



A unique set of interactions between the enzyme and the

cognate DNA

• Direct interaction of GA in 5′-GATATC-3′ with the enzyme

Fig 9.37 Structure of EcoRV embracing a cognate

DNA molecule.



The most striking feature is the distortion of the DNA

• The central TA in 5′-GATATC-3′ is distorted to be positioned for

cleavage, which results in the specificity

• Catalytic activity difference is > 106-fold

Fig 9.38 Distortion of the recognition site. Fig 9.39 Nonspecific and cognate DNA

within EcoRV.

PROTECTION OF THE HOST-CELL DNA


Restriction-modification system

• The host DNA is methylated by methylases for protection

• REs cannot cleave methylated DNA

• For each RE, the host cell produces a corresponding

methylase to methylate the cognate sequence

Fig 9.41 Protection by methylation.

9.4 ATP HYDROLYSIS OF MYOSINS

Myosins comprise a family of ATP-dependent motor proteins

Involved in muscle contraction and a wide range of other

eukaryotic motility processes

Found in all eukaryotes and the human

genome encodes more than 40 different myosins

Catalyze the hydrolysis of ATP

• Produce ADP and inorganic phosphate

• Thermodynamically favorable reaction

• Use the energy to drive the motion of molecules

CHAPTER 9

MYOSIN-ATP COMPLEX STRUCTURE


The ATPase domain structure of the myosin from the soil-living

amoeba Dictyostelium discoideum

• Approximately 750 amino acids

• No significant structural change between the apo form and

complexed form

Fig 9.45 Myosin-ATP complex structure. blue,

no ligands bound; purple, complexed with ATP.

• No hydrolysis observed in the

complexed structure

• Mg2+ is not present in the

enzyme

• All NTPs are present as

NTP-Mg2+ complex

MYOSIN-ATP COMPLEX STRUCTURE


How does the hydrolysis occur?

• Water needs to be activated

• Requires a basic residue or activation by a metal ion

Fig 9.45 Myosin-ATP complex structure. blue,

no ligands bound; purple, complexed with ATP.

The enzyme-ATP complex is

stable

• No basic residue and

nucleophilic water observed

• Conformational change

required for catalysis

THE COMPLEX STRUCTURE WITH A TS-ANALOG


For catalysis, ATPase must stabilize the TS of the reaction

Expected that ATP hydrolysis includes a pentacoordinate TS

The complex structure of the ATPase with VO43-, ADP and Mg2+

• The vanadium atom is coordinated to five oxygen atoms

• Ser236 is positioned to play a role in catalysis

Pentacoordinated transition state of ATPFig 9.46 Myosin ATPase transition state analog.



The proposed mechanism of ATP hydrolysis

• The water molecule attacks the γ-phosphoryl group

• The hydroxyl group of Ser236 mediates the proton transfer

from the water molecule to γ-phosphoryl group

• The ATP serves as a base to promote its own hydrolysis

Fig 9.47 Facilitating water attack.



Conformational change of myosin

• Some residues in the active site moves by ~2 Å

- This helps facilitating the hydrolysis by stabilizing the TS

Fig 9.48 Myosin conformational changes. red,

ATP-bound; blue, TS analog-bound.

• 60 amino acids at the C-

terminus moves by ~25 Å

- This motion is amplified

even more as the C-terminal

domain is connected to

other structures.

THE RATE LIMITING STEP


Slow turnover rate of myosin

• Once per second

• What steps limit the rate of turnover?

Experiment with H218O



Experiment with H218O

• Two or three 18O were observed

→ the hydrolysis reaction is reversible

→ the release of the products (Pi) is rate limiting

Fig 9.48 Reversible hydrolysis of ATP within the myosin active site.



Myosins are examples of P-loop NTPase enzymes

• P-loop is named because it interacts with phosphoryl groups

• P-loop is found in many enzymes involved in ATP-mediated

conformation change

Fig 9.51 Three proteins containing P-loop NTPase domains.

chapter 9: catalytic strategies - sogang ocwocw.sogang.ac.kr/rfile/2014/biochemistry...

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